The coagulation cascade is initiated by binding of the coagulation factor VII(a) (FVIIa) to its cell surface receptor, tissue factor (TF). An aberrant expression of TF is the primary reason for thrombotic disorders associated with various diseases. Proper regulation of TF expression is critical for the maintenance of hemostatic balance and for health in general. The broad and long-term objective of the present proposal is to understand the mechanisms controlling the initiation and regulation of TF-dependent blood coagulation. The majority of TF on cell surfaces exists in a cryptic (coagulant inactive) state. A variety of cellular alterations could transform cryptic TF to coagulant active TF but at present it is unclear even how the coagulant active TF differs from the cryptic form or the mechanics involved in de-encryption of TF. The studies proposed in Aim 1 will examine the conflicting mechanisms that have been proposed to explain TF de-encryption and obtain new data that would provide a better understanding of the process of TF activation. In addition to TF encryption, TF endocytosis and exocytosis could also play a role in the regulation of TF activity at the cell surface but little is known about the molecular processes that regulate TF trafficking. Studies described in Aim 2 will identify and delineate mechanisms that are responsible for TF endocytosis and trafficking. Thus, the specific aims of the proposed studies are (1) delineate the molecular basis for TF activation, particularly in reference to thiol oxidation and phospholipid changes at the outer membrane, and (2) define mechanisms that regulate TF endocytosis and trafficking. The proposed studies will employ a variety of biochemical, molecular and cell biology techniques, including state-of-the art confocal microscopy. Data obtained from the proposed studies will provide new insights towards understanding how TF activity is regulated at cell surfaces and will resolve the recent controversy on TF activation. Overall, the knowledge gained from the proposed studies will be helpful in understanding the pathogenesis of thrombotic disorders and useful for designing better treatment strategies for both hemorrhagic and thrombotic disease 3.